A simple, rapid, and inexpensive microplate tissue culture method suitable for large-scale cytotoxicity screening is described. A variety of esters of helenalin and four dimethylamine adducts of the esters have been synthesized in an effort to evaluate the significance of incorporating increased lipophilic character and/or potential alkylating groups on in vitro cytotoxicity and in vivo antitumor activity. The results indicate that both an increase in lipophilic character and an additional conjugated ester side chain contribute to enhanced cytotoxicity. Maintaining the alpha,beta-unsaturated ketonic moiety is more important than maintaining the alpha-methylene-gamma-lactone moiety, since a high level of cytotoxicity can be obtained when the dimethylamine adduct of the parent molecule is combined with a conjugated ester side chain. Reexamination of the reaction of helenalin with hydrogen chloride in chloroform has led to the formation of the expected mexicanin-A as reported. But instead of neohelenalin accompanying mexicanin-A, 1-epiallohelelnalin and a new oxide were obtained. A new mechanism has been postulated to account for the mode of formation of these compounds. Carolnein and carolenalin, two new guaianolides have been isolated from Helenium automnale K., collected from North Carolina. A preliminary account of the observation for the remarkable effect of epoxidation upon cytotoxicity of the helenalin related derivatives has been made.